We propose to continue our efforts to understand, in genetic and molecular detail, how sex is determined in the fruit fly, Drosophila melanogaster. Experience has shown that the unique features of this system can be effectively exploited to reveal the complexity, diversity, and relatedness of mechanisms controlling developmental processes in higher organisms. Lessons learned in this model system reveal the kinds of phenomena that underlie normal and pathological development of less experimentally tractable organisms like humans and thereby provide insights valuable for improving human health. The remarkable rapidity with which sex determination mechanisms can change provides special opportunities for work in this area to provide insights into the evolution of developmental programs and the nature of genetic change. Methods used in the analysis will include the most modern molecular approaches being applied to many systems of developmental interest; however, the studies benefit as well not only from the more specialized genetic tools available for Drosophila but also from a large set of truly unique genetic tools that have been genetic specifically for the study of fruit fly sex determination over two decades of research from many labs. The project has four general goals: (1) Understand better how and perhaps ultimately why the sex of Drosophila germ cells is determined so differently from that of the soma, and understand better the functional relationships among the many genes known to control fruit fly gametogenesis. (2) Understand how the functioning of genes of the primary sex determination signal is related to their functioning in other developmental pathways and how those relationships are conserved over evolutionary time within this genus. (3) Exploit the unique but previously underutilized advantages of P-element excision and radiation-induced templated gap repair to expand understanding of structure-function relationships for the master sex-determination gene, Sex-lethal, and determine the extent to which conclusions in this area that have been based on in vitro studies and on studies in vivo with transgenes, apply to Sex-lethal in a truly native in vivo context. (4) Explore the possibility of homeostatic negative feedback regulation between Drosophila sex determination and X-chromosome dosage compensation.